L1 - Cell Cycle Control

Function of Cell Division

Function of Cell Division

Embryonic Development Tissue regeneration - to replace naturally dying cells Homeostasis - to repair

The cell cycle

The basis of replication, development and growth Highly conserved across eukaryotes Requires intricate coordination Interphase + M phase Occurs every 24h (ish) Not every cell goes through it

When the cell grows it needs to make more:

Proteins RNA DNA Lipids Organelles

Interphase

The growth phase G1 (Gap 1) + S (Synthesis) + G2 (Gap 2) Over 90% of time spent in this phase The chromosomes are relaxed and long

M phase

The division phase Mitosis + Cytokinesis

Division of cellular components

Most cellular content is divided roughly in two, except for DNA which divided exactly in two

Quiescence

G0 Reversible cell cycle exit Temporarily leaving the cell cycle

Senescence

Permanent cell cycle exit In specialised or damaged cells To prevent cancer by stopping growing entirely

G1 Phase

Gap 1 Growth phase Synthesis of RNA and proteins Where the cycle starts

S phase

Synthesis When DNA replication occurs Cannot finish until all DNA is replicated

G2 phase

Skipped by some cells DNA repair Cell prepares for mitosis At the end of this stage the cell has 2 copies of each 46 chromosomes

Mitotic cells in culture

Round up In order to go through division cells detach and look round rather than flat

Mitosis

DNA is partitioned equally during mitosis with the help of centromeres Results in two diploid daughter cells, identical to the parent Prophase Prometaphase Metaphase Anaphase Telophase

Prophase

Chromosomes condense Spindle aparatus begins to form The two sister chromatids lie togehter, attached at the centromere

Prometaphase

Nuclear envelope breaks down Microtubules contact chromosomes at kinetochores

Metaphase

Chromosomes complete migration to the middle of the cell (equatorial plane) Most highly condensed chromosomes

Anaphase

Sister chromatids separate into daughter chromosomes and are pulled to opposite poles of the spindle apparatus, centromere first End with 92 seperate chromosomes, half near each

Telophase

The nuclear envelope re-forms and chromosomes condense Spindle fibres disappear

Centrosome

The principal microtubule organising centre (MTOC) Duplicated and divided exactly once per cell cycle After duplication the centrosomes migrate to either ends of the cell Most cells only have one centrosome, but some (e.g. cilia) have multiple

The Centrosome Cycle

Centrosome cycle runs at the same time as cell cycle - shares many features

Cell cycle regulation

Cells cannot return to the previous state after a restriction point Checkpoints maintain directionality and as quality control Mostly involving PTMs, small and reversible, covalently attached, needing an enzyme and being veru strong

Kinase

Phosphorylate the target

Phosphatase

Dephosphorylate the target

Cyclin

Non enzymatic protein Levels of cyclin increase and decrease throughout the cell cycle

• Through protein synthesis and cleavage

Cdk

Cyclin dependent kinase A kinase that can phosphorylate targets Requires cyclin to function (only active when bound to cyclin) Can only phosphorylate specific consensus motifs on targets Levels maintained the same throughout the cycle

Maturation promoting factor

First discovered in frogs Fusion of mitotic cells with a cell in any other cycle stage causes premature chromosome condensation - these cells go into mitosis MPF consists of Cyclin and Cdk

Cyclin expression

Different types expressed at different points in the cell cycle Confer substrate specificity for CDK Downstream targets of Cyclin-CDK move the cell cycle forward Levels decrease sharply after the end of the phase - sharp line between phases

Internal influences of cell cycle

Growth DNA replication DNA damage

• Little damage must be repaired

• Lots of damage causes senescence or apoptosis

External influences of the cell cycle

Food Space Communication with organs - tissue damage and developmental stage

• Hormones

Cyclin destruction

Through ubiquitination Causes abrupt decrease in Cdk function after end of phase

Ubiquitin-Proteasome System

Ubiquitin - small PTM, covalently linked Polyubiquitation formed on lysines An entire protein enters the proteasome and amino acids leave These can then be used to make more proteins Ubiquitins open the "lid" of the proteasome so it opens and degrades the protein

APC/C

Anaphase Promoting Complex or Cyclosome In the ubiquitin ligase family (joins ubiquin to something) Key regulator of metaphase to anaphase transition Cyclin is the most important target Coactivators:

• Cdc20 (mitosis to metaphase) -- polybiquinates M cyclin (bound to Cdk) and causes degradation

• Cdh1 (anaphase to the start of S phase)

Cohesin

Holds chromosomes together Cohesin cut by seperase - to go from metaphase to anaphase Seperase held by securin (inhibitory) APC ubiquitinates scurin and so releases separase when all chromosomes are lined up in middle of cell

CKI (CDK inhibitor proteins)

Block Cdk function by covering the protein surface (including active site) or covalently attaching chemical groups Covers the active site of the protein

• Temporary non-covalent interaction

• When DNA damage will stop Cdk from acting and holding in the current cell cycle phase Is broken down by ubiquintation by the SCF complex Two families in mammals:

• CIP/KIP

• INK4

Cdk activation

Cyclin binding is necessary but not sufficient PTMs act to fine regulate Active site of Cdk only fully active when phosphorylated by cyclin activating kinase (CAK)

Wee1 and Cdc25

Wee1 - inhibitory kinase Phosphorylates a neighouring site and blocks the active site Cdc25 - activatory phosphatase Removes the inhibitory phosphate

p21

A Cdk inhibitor Covers the cyclin CDK Regulated by p53

• p53 is often gone in cancer cells

p53

TF to activate p21 transcription In response to DNA damage, p53 is activated by phosphorylation Pauses the cell cycle to repair the damage

Oncogenes

Have a normal function in the cell, when upregulated causes cancer e.g. E2f or Cyclin E

Tumour supressor genes

When missing on not being created causes cancer e.g. Rb

Positive feedback loop (Mitosis)

Ensure a process keeps going Activating the activator and suppressing the suppressor

Cell cycle checkpoints

To ensure there is suitable cell state and environment before proceeding to the next stage Each checkpoint requires a different stimulant and acts through a different Cyclin-Cdk couple G0/G1 = Mitogen stimulation G1/S = Restriction point S/G2 = DNA damage G2/M = Antephase checkpoint M/G1 = SAC

Mitogen stimulation

Mitogen = a molecule that pushes the cell into mitosis Without this it will not enter the cell cycle (will remain in G0) e.g. growth factors

Restriction point

The point at which the cell decides whether to go through with mitosis Mitogen signal acts through G1 and G1/S Cdks - phosphorylating Rb and releasing the Rb targets Rb covers the transcription factor target E2F The uncovering of this causes the transcription of genes needed for cell proliferation

DNA damage mediated arrest

Can occur at any time in the cell cycle After a cascade of signals and sequential phosphorylations, Cdk-cyclin complexes are inhibited - through recruitment of CKIs

Spindle Assembly Checkpoint (SAC)

Activated upon nuclear envelope breakdown To prevent premature segregation of sister chromatids and therefore ensure genome stability Mitotic checkpoint complex (MCC) is the SAC effector molecule, generated at unattached kinetochores

Mitotic checkpoint complex (MCC)

iThe SAC effector molecule, generated at unattached kinetochores Inhibits APC/C in order to prevent premature anaphase and unequal segregation of DNA No longer generated with kinetochores are attached, so APC/C can degrade key substrates

Flow cytometry

Label DNA in samples with dye then fix and sort

Immunohistochemistry (IHC)/Immunofluorescence IF)

Antibodies raised against known mitotic markers Used to observe cell cycle stage

Cytokinesis

Cytoplasmic division Roughly in half

Sister Chromatids

Identical chromosome pairs Often exchange material during interphase

Meiosis

Produce 4 haploid cells from one diploid cell Two cell divisions

Meiosis I

Reduction division stage 2 haploid from 1 diploid Interphase I - replication of DNA Prophase I

• Chromatin coils

• Homologous chromosomes pair up and chromatins intertwine

• Formation of chiasmata - attachments between homologous chromosomes

• Chromosomes begin to move to centre, spindle apparatus begins to form

• Nuclear membrane disappears Metaphase I

• Spindle formation

• Chromosomes align - two centromeres on opposite side of equatorial plane Anaphase I

• Chiasmata disappear

• Homologous chromosomes are pulled by spindle fibres to opposite parts of the cell (one of each pair of autosomes and one of sex chromosomes) Telophase I

• Chromosomes reach opposite ends of cell + slightly uncoil

• Nuclear membrane begins to form

• Cytokinesis (males = equal division, females = unequal - one is polar body)

Meiosis II

Equatorial divison Each haploid cell replicated Interphase II - V brief, no replication Prophase II

• Chromosomes thicken and coil

• Nuclear membrane disappears

• Spindle fibers form Metaphase II

• Spindle fibers pull chromosomes to equatorial plane Anaphase II

• Centromeres split and carry single chromatid to either side Telophase II

• Chromosomes begin to uncoil

• Nuclear membranes formed

• Cytokinesis (males = equally, females = unequal - one becomes polar body(

Spermatogenesis

Constantly occurring Spermatogonia = diploid Mitosis -> primary spermatocyte (diploid) Meiosis I -> 2 secondary spermatocytes (haploid - 23ds) Meiosis II -> 4 spermatids (haploid - 23ss) Spermatids lose cytoplasm and develop tails

Oogenesis

Mostly before birth Oogonia = diploid Mitosis -> primary oocyte (diploid) - during foetal development Held in prophase I until birth, continues when ovulated Meiosis I -> 1 secondary oocyte + 1 polar body (haploid - 23ds) Emerges from follicle and proceeds down fallopian tube w/ polar attached Meiosis II -> 1 mature ovum + 1 polar body (haploid - 23ss)

• Only happens if fertilised by a sperm Polar bodies disintegrate